In the case of investigating kinetics of biomolecules such as proteins, or in investigating interactions between biomolecules, a method for measurement of fluorescence intensity is often employed by labeling the analytes through with the use of phosphorescent or fluorescent dyes. Those fluorescent dyes such as Alexa Fluor, BODIPY FL, Cascade Blue, FITC, Oregon Green, RITC, Texas Red, TRITC, Coumarin Maleimide, Cy Dye, Dansyl Chloride and Dansyl Hydrazine can be used.
By synthesizing non-natural amino acids having functional side chain, by introducing them into proteins at specific positions as the same manner as natural amino acids or by introducing into peptides using peptides synthesizing system, various kinds of functional groups can be introduced into proteins without suppressing their functions. For example, the analyses of kinetics of biomolecules and interactions between them are expected to be carried out simply and accurately, if the incorporation of a non-natural amino acid(s) at specific positions of proteins or the application of a non-natural fluorescent amino acid(s) to the peptides using peptides synthesizing system is performed.
To conduct measurements using conventional instruments, such as a confocal microscope and a microplate reader, subject fluorescent dyes must show absorption wavelengths in visible light range. As widely known fluorescent dyes excitable under visible light may have an extremely large molecular structure, they are inappropriate for fluorescence labeling using protein synthesizing system. Further, when labeling proteins, some of the conventional fluorescent substances have problems in terms of photo stability, easy to inactivate, and high tendency for quenching, during monitoring the kinetics and interactions of the labeled biomolecules.
There is a report on the synthesis of a fluorescent amino acid having an acridine structure (Non-patent document No. 1). Derivatives of a novel acridone dye having a characteristic fluorescence lifetime have been disclosed (Patent document No. 1). Further, patent document No. 1 reported on other acridone dye derivatives, wherein a set of different fluorescent acridone dye derivatives, each of which has a different fluorescence lifetime, is especially useful in multiparameter analysis. However, fluorescent amino acids or acridone dyes derivatives reported in them are fluorescent substances appropriate for use under primarily UV light for excitation. Meanwhile, BODIPY® (Invitrogen), which is a fluorescent substance excitable under visible light and having higher photostability, is commercially available. The compound has a high molar extinction coefficient and high molecular fluorescence yield, thereby being able to emit strong fluorescence, but it is difficult to introduce it into proteins, because it has large-size side chain and it may break the higher-order structure of the proteins.
[Non-patent document No. 1] Helvetica Chimica Acta., 86, 3326 (2003)
[Patent document No. 1] JP 2005-500406 A